In the past year we have continued to focus our studies on 1) the development of a model that may provide insights into mechanisms involved in autoimmune reactions in skin and in the maintainance of tolerance to epidermally-derived proteins and 2) the identification of the specific precursors of Langerhans cells.&lt;BR&gt;&lt;BR&gt;The major project that we are pursuing involves the characterization of a model of skin autoimmunity and peripheral immunological tolerance induction. We have developed transgenic mice that have a K14-ovalbumin (K-14 OVA) encoding gene. Some of the mice express the OVA on the epithelial cell surfaces (K14-mOVA) while others express soluble OVA (K14-sOVA). We are studying these mice and have also crossed these mice with those that have a TCR transgene that recognizes ovalbumin in association with H-2b (OT-I). These mice have the TCR for ovalbumin and express ovalbumin in the epidermis but have no apparent disease. We are also using the K-14 OVA mice as targets for immunological reactions in the skin. When T cells from OT-I mice are injected into either the single Tg mice or the double Tg mice their role in causing inflammatory skin lesions has been assessed. We have found that the OT-I CD8+ T cells induce a GvHD-like disease in the single Tg (k14-mOVA) mice....this is characterized by swelling of the feet from days 6-14 and development of redness and scaling of the skin within 7 days. The mice lose weight and die between weeks 2 and 3. The mice probably die because of a mucositis (tongue and esophagus)-they are unable to eat properly. In sharp contrast, the double Tg (K14-mOVA X OT-I) mice, despite their expressing OVA in the skin, are unaffected when the OT-I CD8+ T cells are injected into them. One of the aims of our current studies is to better understand why there is tolerance in these double Tg mice. We have been unable to clearly delineate a CD4+CD25+ T regulatory cell in the double Tg mice but have identified a CD3+ double negative cell population in these mice and are focussing on the possibility that they are somehow serving as regulatory cells. We have found that in single transgenic (K14/mOVA no.3 mice that are low producers of OVA(K14-mOVAlow) there is tolerance to the adoptively transferred OT-I cells. We have found that IL-15 can enhance the activity of the injected OT-I cells in these mice and induce GvHD. We have also started focussing on K14-OVA mice that produce soluble OVA (K14-sOVA). When OT-I cells are injected into these mice, almost all mice die after 5-7 days. and this is due to an acute mucositis and esophagitis. We are currently studying the mechanism by which this destruction takes place.&lt;BR&gt;&lt;BR&gt; During the past year we found that K14-sOVA crossed with OT-I mice die within 15-21 days of birth. The cause of death is probably due autoreactivity of OT-I cells that have not been deleted by the thymus. Recent studies have shown that we can obviate death by injecting the relevant peptide (SIINFEKL) on days 5 and 9 to these double transgenic mice. Thus, while all of these double transgenic mice die within 21 days if PBS is injected on days 5 and 9 of birth, about 50% survive if 10 micrograms of peptide is injected on days 5 and 9. Furthermore, if 100 micrograms is injected on days 5 and 9 an even higher percentage of these double transgenic mice survive beyond day 21. The mechanism by which these peptides protect the double Tg mice is currently being studied- early findings suggest that these is a depletion of CD8 cells in these mice and these CD8 T cells express many fewer CD8 molecules than do the double Tg mice that die. There also seems to be downregulation of the V alpha chain of the TCR on these CD8+ T cells. Furthermore, CD8 T cells from healthy (treated) mice were anergic and could not be activated by exogenous IL-2. A block in IL-2/IL-7 signaling via the STAT5 pathway provided the basis for low surface expression of the CD8 coreceptor and failure of IL-2 to break CD8 T cell anergy. Thus, the soluble TCR ligand triggered multiple tolerance mechanisms in these sOVA/OT-I mice, making this treatment approach a potential paradigm for modulating human autoimmune diseases. Finally, in trying to determine the target for the OT-I cytotoxic T cells in all of these models, we have found that in conditional knockout mice (for Langerhans cells [LC]), even in the absence of LC, GvHD occurs. We have also created chimeric mice in which the antigen-presenting cells (APC) cannot present OVA peptides to CD8+ cells because the APC are beta 2 microglobulin deficient and these mice also develop GvHD when OT-I cells are adoptively transferred. In vitro studies have demonstrated that keratinocytes, in the absence of LC, can present antigen to the OT-I cells.